Construction Of Near-Infrared Xanthene Dyes For Bioimaging

Recently,with the development of fluorescence imaging instrument(such as single-photon and two-photon laser confocal fluorescence microscopes,CT or X-ray compatible live fluorescence imaging systems,super-resolution fluorescence microscopes),fluorescence imaging technology has become a powerful tool for visualizing biological molecules in living systems and exploring their biological roles due to the characteristics of damage-free,high sensitivity and real-time spatial imaging.Near-infrared light(650-900 nm)has the advantages of deep tissue penetrability,minimal photodamage to biological samples and can avoid the interference from autofluorescence of biological molecules.Therefore,near-infrared fluorescent dyes have great application prospects in the field of biological tissue and living animal imaging which attracted great attention of researchers.Traditional xanthene dyes,such as Rhodamines,have become one of the most widely used dyes due to the advantages of high fluorescence quantum yield,large molar extinction coefficient,excellent photostability and biocompatibility.However,their relatively short absorption and emission wavelengths(located in the visible light region)limit their applications in biological and tissue imaging.Therefore,improving the emission wavelength of traditional xanthene dyes by chemical methods to make them red-shift to near-infrared region(650-900 nm)or near-infrared II region(900-1700 nm)has become a hot research topic.Rhodamine,Rhodol,Fluorescein are important members of xanthene family dyes,by modifying the structure of the dyes,their absorption and emission spectra have been extended to the entire near-infrared region.The general strategy can be summarized as(1)modification of the amine groups;(2)extending π-conjugation of the xanthene ring;(3)replacing the central oxygen atom with other elements;(4)introducing electron-withdrawing groups to the meso-position.Although,researchers have developed a large number of near-infrared xanthracene dyes by the above strategies and successfully applied them to the identification and labeling of biomolecules.There are still some problems,for example,the introduction of electron-withdrawing groups at the 9-position to increase the wavelength of xanthene fluorescent dyes is still in its infancy compared with other methods and there is still plenty of room for further explorations.Secondly,most of the near-infrared fluorescent dyes constructed by expanding the xanthracene ring π-conjugate strategy are rhodamine derivatives,while,few studies on rhodol dyes which limited the application of rhodol dyes in biological imaging.The main purpose of this thesis is solving the above-mentioned problems,synthesizing some new near-infrared xanthracene fluorescent dyes by designning the molecular structure and further exploring its applications in biological imaging and fluorescent probes.Based on this idea,this article mainly carried out the following three parts:In Chapter 2,we present a benzothiazole-functionalized rhodamine dye BTP as a red-emission fluorescent dye platform for bioimaging applications.Due to the electron-withdrawing nature of the meso-substituted benzothiazole unit,BTP exhibited a large red-shift in absorption and emission wavelengths compared to classic rhodamines.Interestingly,BTP could not only behave like a molecular rotor to fluorescently respond to viscosity changes,but also specifically target lysosomes and light up them assisted by a lysosomal viscous microenvironment.Furthermore,based on the BTP platform,we developed its ‘dihydro’ derivative,i.e.,HBTP,and evaluated its sensing performance to reactive oxygen species(ROS).The obtained results showed that HBTP is a highly selective fluorescent probe for sensing endogenous peroxynitrite(ONOO-)with quite rapid fluorescence off–on response and high sensitivity.In Chapter 3,we synthesized three NIR fluorescent dyes Si BM1-3 by introducing benzimidazole derivative at the 9-position of silicon-rhodamine.The emission wavelength of Si BM1-3 are above 700 nm due to the electron-withdrawing effect of benzimidazole.Moreover,Si BM1-3 also have excellent photostability and chemical stability and can selectively enter the lysosome.Base on this platform,we further synthesized its "dihydro" derivative HSi BM1-3 and evaluated the performance for sensing reactive oxygen species(ROS).The experimental results showed that HSi BM3 could specifically,fastly(within 2 s)and sensitively(detection limits is 13.5 n M)react with peroxynitrite(ONOO-)to produce Si BM3.What’s more,HSi BM3 not only used to image exogenous and endogenous ONOO-in cells and mice,but it also enable visualization of drug-induced liver damage.In Chapter 4,we present a new rhodol-hemicyanine fluorescent dye Rd H by combining the structures of a rhodol dye and a hemicyanine group which displayed a longer emission wavelength(688 nm)and a large Stokes shift(Δλ≈110 nm)due to the expanded π-conjugation and the strong intramolecular charge transfer(ICT)property.More important,Rd H can be used as a selective,rapid-response,ratiometric,and reversible fluorescent probe for intracellular GSH(t1/2= 89 ms,Kd= 1.42 m M).The imaging assays in living cells revealed that Rd H could be used to real-time monitor GSH dynamics in A549 cells under a laser scanning confocal microscope by ratiometric fluorescence changes.